Peptide display to antigen presenting cells using lipid vehicle

Abstract

The present disclosure relates to specific delivery of a lipid-peptide conjugate to immune cells ex vivo or in vivo for decreasing or increasing an immune response against therapeutically relevant antigens. The lipid-peptide-antigen is comprised of a peptide, a lipid, and a functional group that is degraded in a biological environment within cells to release the peptide for MHC presentation and provides a more efficient presentation of antigen epitopes by antigen presenting cells than peptide epitopes alone.

1 . A lipid vehicle comprising at least one lipid-peptide conjugate,
 the lipid-peptide conjugate comprising a lipid moiety and a peptide moiety covalently conjugated by a linker,   wherein the lipid moiety is selected from the group consisting of cholesterol, PEGylated phospholipid, and any combination thereof, and   wherein the peptide moiety is an epitope of a therapeutically relevant antigen.   
     
     
         2 . A lipid vehicle comprising at least one lipid-peptide conjugate and a liposome,
 the lipid-peptide conjugate comprising a lipid moiety and a peptide moiety covalently conjugated by a linker that comprises a disulfide bond,   wherein the peptide moiety is an epitope of a therapeutically relevant antigen, and   wherein the liposome has a diameter of about 50-900 nm.   
     
     
         3 . The lipid vehicle of  claim 1 , wherein:
 (a) the peptide moiety has a length of between 6 and 10 amino acids, between 8 and 40 amino acids, between 8 and 30 amino acids, between 8 and 20 amino acids, or between 8 and 15 amino acids;   (b) the linker is biodegradable, is redox sensitive, is hydrolyzed at pH below 7, below 6, or below 5, and/or is self-immolative; and/or   (c) the lipid-peptide conjugate has a structure according to Formula (I):
   Lipid-S—S-Peptide  (I).
 
   
     
     
         4 - 5 . (canceled) 
     
     
         6 . The lipid vehicle of  claim 1 , wherein:
 (a) the lipid-peptide conjugate has a structure according to Formula (II):   
       
         
           
           
               
               
           
         
         (b) the lipid-peptide conjugate has a structure according to Formula (III): 
       
       
         
           
           
               
               
           
         
         (c) the lipid-peptide conjugate has a structure according to formula (IV):
   Lipid-X-Peptide  (IV),
 
 wherein X is a hydrolysable functional group selected from the group consisting of ester, thioester, orthoester, ketal, and imine: 
 
         (d) the peptide moiety is conjugated to the lipid moiety by reaction with a compound having the structure of formula (V): 
       
       
         
           
           
               
               
           
         
         
           optionally wherein X is a linker atom or a linker molecule; 
         
         (e) the lipid-peptide conjugate has a structure according to formula (VI): 
       
       
         
           
           
               
               
           
         
         
           wherein Y is C═O, C═S, or C═NH; 
           X is a C 1 -C 10  alkyl or branched C 1 -C 10  alkyl; 
           m is an integer selected from 0 to 100; 
           Z is NH, O, S, or CH 2 ; and 
           k is an integer selected from 0 to 5; 
         
         (f) the peptide moiety is conjugated to the lipid moiety by reaction with a compound having the structure of formula (VII): 
       
       
         
           
           
               
               
           
         
         
           wherein X and Y are each independently selected from the group consisting of C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; 
           m is an integer selected from 0 to 10; 
           n is an integer selected from 0 to 10; and 
           R is hydrogen, SO 3 H, C 1 -C 10  alkyl, or branched C 1 -C 10  alkyl; 
         
         (g) the peptide moiety is conjugated to the lipid moiety by reaction with a compound having the structure of formula (VIII): 
       
       
         
           
           
               
               
           
         
         
           wherein X and Y are each independently selected from the group consisting of C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; 
           j is an integer selected from 0 to 10; 
           k is an integer selected from 0 to 10; and 
           l is an integer selected from 0 to 10; 
         
         (h) the lipid-peptide conjugate has a structure according to formula (IX): 
       
       
         
           
           
               
               
           
         
         
           wherein X and Y are each independently selected from the group consisting of C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; 
           j is an integer selected from 0 to 10; 
           k is an integer selected from 0 to 10; 
           l is an integer selected from 0 to 10; and 
           R 1  is a single bond or selected from the group consisting of hydrogen, NH 2 , COOH, CONH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; 
         
         (i) the lipid-peptide conjugate has a structure according to formula (X): 
       
       
         
           
           
               
               
           
         
         
           wherein X and Y are each independently selected from the group consisting of C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; and 
           R is hydrogen, SO 3 H, C 1 -C 10  alkyl, or branched C 1 -C 10  alkyl; 
         
         (j) the lipid-peptide conjugate has a structure according to formula (XI): 
       
       
         
           
           
               
               
           
         
         
           wherein X and Y are each independently selected from the group consisting of C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; 
           j is an integer selected from 0 to 10; 
           k is an integer selected from 0 to 10; 
           l is an integer selected from 0 to 10; 
           R 1  is a single bond or selected from the group consisting of hydrogen, NH 2 , COOH CONH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; and 
           R 2  and R 3  are each independently selected from the group consisting of hydrogen, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, or cyclized C 3 -C 10  alkyl: 
         
         (k) the lipid-peptide conjugate has a structure according to formula (XII): 
       
       
         
           
           
               
               
           
         
         
           wherein X and Y are each independently selected from the group consisting of C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; 
           l is an integer selected from 0 to 10; 
           m is an integer selected from 0 to 100; 
           R 1  is a single bond or selected from the group consisting of hydrogen, NH 2 , COOH, CONH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; and 
           R 2  and R 3  are each independently selected from the group consisting of hydrogen, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, and cyclized C 3 -C 10  alkyl; 
         
         (l) the lipid-peptide conjugate has a structure according to formula (XIII): 
       
       
         
           
           
               
               
           
         
         
           wherein X and Y are each independently selected from the group consisting of C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; 
           l is an integer selected from 0 to 10; 
           j is an integer selected from 0 to 100; 
           k is an integer selected from 0 to 10; 
           R 1  is a single bond or selected from the group consisting of hydrogen, NH 2 , COOH, CONH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; and 
           R 2  is hydrogen, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, or cyclized C 3 -C 10  alkyl; 
         
         (m) the lipid-peptide conjugate has a structure according to formula (XIV): 
       
       
         
           
           
               
               
           
         
         
           wherein X is S, C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, or O; 
           l is an integer selected from 0 to 10; and 
           m is an integer selected from 0 to 10; 
         
         (n) the lipid-peptide conjugate has a structure according to formula (XV): 
       
       
         
           
           
               
               
           
         
         
           wherein X and Y are each independently selected from the group consisting of C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; 
           j is an integer selected from 0 to 100; 
           k is an integer selected from 0 to 10; and 
           R is hydrogen, SO 3 H, C 1 -C 10  alkyl, or branched C 1 -C 10  alkyl; or 
         
         (o) the lipid-peptide conjugate has a structure according to formula (XVI): 
       
       
         
           
           
               
               
           
         
         
           wherein X and Y are each independently selected from the group consisting of C═O, C═S, C═NH, C 1 -C 10  alkyl, branched C 1 -C 10  alkyl, NH, S, and O; and 
           m is an integer selected from 0 to 10. 
         
       
     
     
         7 - 21 . (canceled) 
     
     
         22 . The lipid vehicle of  claim 1 , wherein the lipid vehicle has a net positive charge. 
     
     
         23 . The lipid vehicle of  claim 1 , wherein:
 (a) the lipid vehicle comprises at least two distinct lipid-peptide conjugate species, at least 5 distinct lipid-peptide conjugate species, at least 10 distinct lipid-peptide conjugate species, or at least 50 distinct lipid-peptide conjugate species, wherein the at least two distinct lipid-peptide conjugate species comprise distinct epitopes for the same antigen or different antigens;   (b) the lipid vehicle comprises at least one cationic lipid selected from the group consisting of: hydrogenated soybean phosphatidylcholine (HSPC), stearylamine (SA), lauryltrimethylammonium bromide, cetyltrimethylammonium bromide, myristyl trimethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), 36-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), 1,2-ditetradecanoyl-3-trimethylammonium-propane (DMTAP), 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP derivatives such as 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane and 1,2-dihexadecanoyl-3-trimethylammonium-propane, 1,2-di-(9Z-octadecenoyl)-3-dimethylammoniumpropane (DODAP) and DODAP derivatives such as 1,2-ditetradecanoyl-3-dimethylammonium-propane, 1,2-dihexadecanoyl-3-dimethylammoniumpropane, and 1,2-dioctadecanoyl-3-dimethylammonium-propane, 1,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-c-(4′-trimethylammonium)-butanoyl-sn-glycerol (DOTB), dioctadecylamideglycylspermine, SAINT-2, polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido) ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GL67TM, polyLysine lipid conjugates, and polyArginine lipid conjugates;   (c) the lipid vehicle adheres to antigen presenting cells in blood, wherein preferably the peptide moiety is released from the lipid vehicle within 30 days, within 20 days, within 10 days, or within 2 days; wherein preferably less than 20% of the peptide is released from the lipid vehicle after 24 hours, and at least 70% of the peptide is released from the lipid vehicle within 20 days under physiological conditions: wherein preferably when administered to a subject, the lipid vehicle is internalized by antigen presenting cells at least 3 times, at least 10 times faster, at least 30 times faster, or at least 100 times faster than the unconjugated peptide;   (d) the lipid vehicle has a diameter of about 50-500 nm or about 100-200 nm;   (e) the lipid vehicle comprises one or more of: HSPC, DSPC, DPPC, cholesterol, POPC, DOPC, DSPE-PEG2000, DSPE-PEG5000, DOPE-PEG2000, DSTAP and DOTAP chloride;   (f) the lipid vehicle comprises a mixture of HSPC, cholesterol and DSPE-PEG2000; or   (g) the lipid vehicle comprises a mixture of POPC, cholesterol, DOTAP chloride and DOPE-PEG2000.   
     
     
         24 - 28 . (canceled) 
     
     
         29 . The lipid vehicle of  claim 1 , further comprising an immunomodulatory agent, optionally an immunostimulating compound. 
     
     
         30 . The lipid vehicle of  claim 29 , wherein:
 (a) the immunostimulating compound is a ligand that binds to an intracellular protein and/or a receptor, said receptor being selected from the group consisting of TLR3, TLR4, TLR7, TLR8, TLR9, and STING, preferably wherein said receptor is selected from the group consisting of TLR3, TLR4, TLR7, and TLR9, and further preferably wherein said receptor is TLR7;   (b) the immunostimulating compound is selected from the group consisting of: polyinosinic:polycytidylic acid (poly I:C), Polyadenylic-polyuridylic acid (poly A:U), poly I:C-poly-L-lysine (poly-ICLC), poly-ICR, CL264, N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteine-(S)serine-(S)lysine 4 (Pam3Cys), Monophosphoryl lipid A (MPLA) and other lipopolysaccharides, alphagalactosylceremaide (αGC), Propirimine, Imiquimod (R837), resiquimod (R848), Gardiquimod, TMX, TMX201, TMX202, R850, R851, 852A, S-27610, 3M-002 (CL075), 3M-003, 3M-005, 3M-006, 3M-007, 3M-012, 3M-13, 3M-031, 3M-854, CL097, CL264, IC-31, Loxoribine and other imidazoquinolines, ssPolyU, sotirimod, Isatoribine, ANA975, SM360320, R1354 single stranded or double stranded RNA, ORN 02 (5′-UUAUUAUUAUUAUUAUUAUU-3′), ORN 06 5′-UUGUUGUUGUUGUUGUUGUU-3′, CpG-ODN DSLIM, AVE 0675, CpG B oligodeoxynucleotide 1018, LPS, AZD 1419, ODN 1982, CpG B ODN 2006, IMO 2125, CpG A ODN 2216, CpG A ODN 2336, CpG 2395, CpG ODN 7909, CpG 10101, CpG ODN AVE0675, CpG ODN HYB2093, CpG ODN HYB2055, CpG-ODN IMO 2125, CpG C ODN M362, Tolamba (Amb al ragweed allergen with covalently linked CpG B class ODN 1018), Heplisav, 10181SS IM02055 IRS954, flagellin, muramyl dipeptide, saponins such as QS21,  Leishmania  elongation factor, SB-AS4, threonyl-muramyl dipeptide, L18-MDP, mifamurtid, and OM-174;   (c) the immunostimulating compound is selected from the group consisting of: monophosphoryl lipid A (MPLA), Imiquimod (R837), resiquimod (R848), Gardiquimod, TMX, TMX201, TMX202, Loxoribine, sotirimod, Isatoribine, SM360320, CpG B oligodeoxynucleotide 1018, AZD 1419, ODN 1982, CpG B ODN 2006, LPS, IMO 2125, CpG A ODN 2216, CpG A ODN 2336, CpG 2395, CpG ODN 7909, CpG 10101, CpG ODN AVE0675, CpG ODN HYB2093, CpG ODN HYB2055, CpG-ODN IMO-2125, CpG C ODN M362, Tolamba (Amb al ragweed allergen with covalently linked CpG B class ODN 1018), Heplisav, QS21, and OM-174; or   (d) the immunomodulatory agent is an immunosuppressive compound, wherein preferably the immunosuppressive compound is selected from the group consisting of: vitamin D3 (1,25-dihydroxyvitamin D3) and its related synthetic or national analogues, retinoic acid (all-trans and 9-cis retinoic acid) and its related synthetic or natural analogues, Betamethasone hemisuccinate, Dexamethasone palmitate, Dexamethasone phosphate, Limethasone, Methylprednisolone hemisuccinate, Prednisolone palmitate, and Prednisolone phosphate.   
     
     
         31 - 33 . (canceled) 
     
     
         34 . The lipid vehicle of  claim 1 , further comprising a targeting moiety selected from the group consisting of peptides, antibodies, antibody fragments, and nucleotides, wherein preferably the targeting moiety has an affinity against a target selected from the group consisting of: DCIR, CD4, CD8, CD25, CD69, CD45, Ly6C, CD40, CD80, CD86, CD11b, CD11c, CD115, F4/80, CD68, CD14, CD16, CD64, CD163, CD68, CD19, CD1c, CD83, CD141, CD209, MHCII, and Gr1. 
     
     
         35 . A pharmaceutical composition comprising the lipid vehicle of  claim 1 . 
     
     
         36 . The pharmaceutical composition of  claim 35 , further comprising at least one immune effector cell, optionally wherein the immune effector cell is a T cell or a NK cell. 
     
     
         37 . A method of treating cancer by stimulating or enhancing a tumor antigen-specific immune response in a human subject, comprising administering the pharmaceutical composition of  claim 35  to the subject in need thereof. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . A method of in vitro training of T cells, comprising the steps of:
 (a) incubating monocytes and/or immature dendritic cells with the lipid vehicle of  claim 1 , thereby obtaining matured dendritic cells;   (b) mixing the matured dendritic cells with immature T cells and incubating for sufficient time to activate the T cells, resulting in clonal expansion thereof; and   (c) optionally, repeating steps (a) and (b) until a sufficient amount of reactive T cells have been obtained, further optionally wherein the steps are repeated 2-3 times.   
     
     
         41 . A method for preparing antigen-presenting cells (APCs), the method comprising:
 (a) contacting a population of monocytes, immature dendritic cells, and/or dendritic cells with a plurality of lipid vehicles in a medium under suitable conditions for the monocytes, immature dendritic cells, and/or dendritic cells to internalize one or more of the lipid vehicles, wherein each of the lipid vehicles comprises a plurality of lipid-peptide conjugates, each such conjugate comprising a lipid moiety and a peptide moiety covalently conjugated by a linker, and wherein each peptide moiety comprises a peptide fragment of an antigen; and   (b) incubating the monocytes, immature dendritic cells, and/or dendritic cells in the presence of one or more cytokines and/or growth factors under suitable conditions to induce differentiation of the monocytes, maturation of the immature dendritic cells, and/or expansion of the dendritic cells, thereby to prepare a population of APCs.   
     
     
         42 - 51 . (canceled) 
     
     
         52 . A modified immune cell comprising one or more lipid vehicles of  claim 1 , surface-associated with an immune cell. 
     
     
         53 - 55 . (canceled) 
     
     
         56 . A pharmaceutical composition comprising at least one modified immune cell of  claim 52 , and further comprising a pharmaceutically acceptable solution, carrier, excipient, or stabilizer. 
     
     
         57 . A method for treating or preventing a disease or disorder by stimulating, enhancing, or modulating an immune response in a subject in need thereof, the method comprising administering to the subject a composition comprising the modified immune cell of  claim 52 , wherein the immune cell is a dendritic cell or a T cell. 
     
     
         58 . (canceled) 
     
     
         59 . A method for treating or preventing a disease or disorder by stimulating, enhancing, or modulating an immune response in a subject in need thereof, the method comprising: administering to the subject a first composition comprising the lipid vehicle of  claim 1 ; and/or a second composition comprising a modified immune cell comprising one or more lipid vehicles of  claim 1  surface associated with the immune cell. 
     
     
         60 - 63 . (canceled) 
     
     
         64 . The lipid vehicle of  claim 1 , wherein the antigen is associated with a disease, optionally wherein the disease is allergy, autoimmune disease, infectious disease or cancer.